Flock House Virus (FHV) is a simple RNA insect virus that infects drosophila line 1 (DL1) cells. The capsid contains 180 copies of a single protein type arranged with T=3 quasi symmetry. The virus genome is split into two RNA molecules; RNA1 encodes a RNA directed RNA polymerase and a small protein that inhibits the cellular RNAi activity against FHV replication; RNA2 encodes the 407 amino acid capsid protein alpha that assembles into capsids and then undergoes auto-catalytic clevage into beta (residues 1-363) and gamma (364-407), an event required for infectivity. FHV is the simplest non enveloped animal virus, but it shares entry properties with reovirus, blue tongue virus poliovirus and adenovirus. The relationship between genotype and phenotype is extremely well defined. During the first four years of support we have assembled reagents and techniques that allow us to study events associated with virus entry in vivo and in vitro. This provides an exceptional opportunity to understand non enveloped virus during the second half of this MERIT award. Specific Aims: (1) Image FHV (tetra-cys containing) VLP entry into live DL1 cells with the fluorescent probes FIAsH and ReAsH. Confocal imaging will allow the determination of the precise time course of entry without infection, as VLPs do not contain the viral genome. This will define the time points for study with high pressure freezing and electron tomography. (2) Time course studies of high pressure frozen, thick sections will be performed with electron tomography to follow the entry pathway of virus particles, the location of uncoating and the translocation of RNA across endosomal membranes. (3) Studies with VLPs depositing tetra-cys modifies gamma peptides, but not infecting cells, will be investigated to determine exactly where gamma peptides are deposited and what membranes are effected. The studies will be performed with ReAsH, allowing fluorescence and high resolution EM studies of the DL1 cells. (4) A cryoEM reconstructionis being performed on the eluted particles of FHV, corresponding to a disassembly intermediate. (5) Features of the gamma peptide required for membrane alteration and infection will be determined with the gamma in trans procedure discovered during the last period of support. (6) The putative lachesin receptor for FHV will be used as a reagent to understand virus particle destabilization in vitro. (7) Crystallographic studies will be performed with the RNA polymerase to establish a structural basis for its multi-functionality.